The tightly regulated homeostatic mechanisms between hepatocyte proliferation and apoptosis that exist in normal liver tissue are disrupted in many liver diseases and during hepatocarcinogenesis. The transforming growth factor beta (TGFbeta) signaling pathway is a central component of the mechanisms by which cell growth and apoptosis are regulated in the liver. The long-term objective of this project is to understand the molecular mechanism by which TGFbetaa regulates hepatocyte proliferation and apoptosis. TGFbeta is a potent inducer of hepatocyte apoptosis both in vivo and in vitro. The apoptotic activity of TGFbeta can be antagonized by the action of insulin and its downstream signaling molecules. This proposal is designed to understand how TGFbeta pathway and insulin pathway cross talk to regulate the sensitivity of hepatocytes to TGFbeta-induced apoptosis. Smad proteins are critical mediators of TGFbeta signaling. In the absence of ligand, Smad2 and Smad3 are located in the cytoplasm. Upon binding of TGFbeta to its receptors and the subsequent activation of the receptor serine/threonine kinases, Smad2 and Smad3 become phosphorylated by the receptor kinases and then translocate to the nucleus, where they can form heteromeric complexes with Smad4 and activate transcription of TGFbeta responsive genes. Among these Smad proteins, Smad3 has been shown to mediate TGFbeta-induced apoptosis. In a search for molecules that interact with Smad3, the P.I. identified Akt, a critical molecule functioning in the cell survival pathway downstream of insulin. Akt can directly interact with Smad3, and this interaction is regulated by TGFbeta and insulin. Because expression of an activated Akt results in protection of cells from TGFbeta-induced apoptosis, we hypothesized that Akt may protect cells from TGFbeta-induced apoptosis by interacting with Smad3 and preventing it from mediating the apoptotic signals of TGFbeta. The following specific aims are designed to further characterize the interaction between Akt and Smad3 and uncover the physiological significance of this interaction. The specific aims are: 1) Mapping the amino acid residues in Akt and Smad3 required for their interaction. 2) Analysis of the role of the Smad3- Akt interaction in protection from TGFbeta-induced apoptosis. 3) Analysis of the molecular mechanism by which Akt inhibits TGFbeta-induced apoptosis. In particular, we will investigate whether Akt inhibits Smad3 through affecting its phosphorylation or through physical sequestration. These studies will allow us to have a better understanding of how TGFa and insulin signaling pathways cross talk to regulate the sensitivity to TGFbeta-induced apoptosis. Since this cross talk plays an important role in the control of liver size and disruption of this highly regulated process can result in liver diseases and hepatocarcinogenesis, these studies may contribute to a better understanding of the maintenance of normal liver homeostasis and the cause of liver diseases.